1. Arabidopsis ABF3 and ABF4 Transcription Factors Act with the NF-YC Complex to Regulate SOC1 Expression and Mediate Drought-Accelerated Flowering 
Keumbi Hwang, Hendry Susila, Zeeshan Nasim, Ji-Yul Jung, Ji Hoon Ahn 
Molecular Plant 
Volume 12, Issue 4, Pages (April 2019)
DOI: /j.molp
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2. Figure 1
Late-Flowering Phenotype of abf3 abf4 Mutants at 23°C under LD Conditions.
(A) Total leaf number at flowering for abf mutants (see Methods for further information on box plots).
(B) Late-flowering phenotype of abf3 abf4 and abf2 abf3 abf4 mutants grown at 23°C under LD conditions. The wild-type plants and abf mutants in this photo were sown at the same time.
(C) Confirmation of HA-ABF3-SRDX protein expression in individual 35S::3×HA:ABF3:SRDX transgenic lines. Protein levels were measured at ZT8 from 8-day-old seedlings. Levels of HA-ABF3-SRDX in wild-type plants were set to 1.
(D) Total leaf number of 35S::3×HA:ABF3:SRDX transgenic lines grown at 23°C under LD conditions.
(E) Total leaf number of abf3 abf4 mutants grown on MS medium supplemented with 5 μM ABA (left) or grown in soil (right). ABA (20 μM) was provided three times a week until the soil-grown abf3 abf4 mutants bolted.
Asterisks represent significant differences derived from two-tailed Student's t-tests (ns, not significant; *P ≤ 0.05, **P ≤ 0.01, ****P ≤ ).
Molecular Plant  , DOI: ( /j.molp )
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3. Figure 2
Expression Levels of ABF3 and ABF4 during Early Seedling Development.
(A) The mRNA levels of ABF3 and ABF4 from 2 to 12 days after germination (DAG). Seedlings were grown at 23°C under LD conditions, and each sample was collected at ZT8. mRNA levels of ABF3 and ABF4 at 2 DAG were set to 1. The mRNA levels of ABI3, ABI4, ABI5, and AP1 were analyzed for comparison.
(B) Validation of the native promoter-driven HA-tagged ABF transgenic plants. ABA solution (20 μM) was sprayed on 8-day-old seedlings at ZT0, and HA-ABF3 and HA-ABF4 protein levels were measured at ZT4 to assess ABF3 and ABF4 promoter activities. Levels of HA-ABF3 and HA-ABF4 were set to 1.
(C) Expression of HA-tagged ABF3 and HA-tagged ABF4 during early seedling development in pABF3::3×HA:ABF3 abf3 and pABF4::3×HA:ABF4 abf4 plants under LD conditions. Levels of HA-ABF3 and HA-ABF4 at 2 DAG were set to 1.
(D) GUS histochemical analyses of pABF3∷GUS and pABF4::GUS plants from 2 DAG to 12 DAG. Scale bar = 0.1 cm.
(E and F) Diurnal expression levels of transgenic ABF3 and ABF4 mRNA (E) and HA-tagged ABF3 and HA-tagged ABF4 (F) in pABF3::3×HA:ABF3 abf3 and pABF4::3×HA:ABF4 abf4 plants under LD conditions. Levels of HA-ABF3 and HA-ABF4 at ZT0 were set to 1.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Author Terms and Conditions

4. Figure 3
Effects of Misexpression of ABF2, ABF3, or ABF4 in the Shoot Apex and the Vasculature on Flowering Time.
(A and B) Validation of misexpression of ABF2, ABF3, and ABF4 (A) in the vasculature of pSUC2::ABF plants (left panels) and in the shoot apical region of pFD::ABF plants (right panels). L, leaf; R, root; A, shoot apex. (B) Flowering time of pSUC2::ABF plants and pFD::ABF plants at 23°C under LD conditions.
(C) Flowering time of 35S::ABF3 and 35S::ABF4 plants at 23°C under LD conditions. For qPCR, data obtained from three biological replicates are shown.
Asterisks represent significant differences derived from one-way ANOVA followed by Dunnett's multiple comparisons tests (ns, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Author Terms and Conditions

5. Figure 4
Identification of SOC1 as a Downstream Target of ABF3 and ABF4.
(A) The Venn diagram (left) shows the 122 flowering-time genes bound by both ABF3 and ABF4 (Song et al., 2016a). The second Venn diagram (right) highlights 11 out of the 122 common target genes of ABFs that were significantly induced upon ABA treatment in wild-type Col-0 plants (GSE65016 and GSE66737) (Zhan et al., 2015). +, floral promoters; −, floral repressors.
(B) SOC1 RNA-seq peaks and ChIP-seq peaks. For the RNA-seq peaks, induction under mock treatment (red) and ABA treatment (blue) is depicted. For the ChIP-seq peaks, the binding of ABF3 and ABF4 to the upstream promoter region under mock (ethanol) treatment (red) and ABA treatment (blue) is shown.
(C) SOC1 mRNA levels in 8-day-old abf mutants grown at 23°C (ZT8) under LD conditions determined by qPCR. Asterisks represent significant differences (ns, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Author Terms and Conditions

6. Figure 5
ABF3 and ABF4 Associate with the Promoter of SOC1 and Induce Its Expression.
(A) Diurnal expression levels of SOC1 mRNA in wild-type plants and abf3 abf4 mutants determined by qPCR. Open and closed boxes below indicate day and night, respectively.
(B) Different levels of SOC1 mRNA induction by 20 μM ABA treatment in 8-day-old wild-type plants and abf3 abf4 mutants. Wild-type plants and abf3 abf4 mutants were treated with ABA at ZT0, and SOC1 mRNA levels were measured every 4 h. SOC1 mRNA levels in mock-treated wild-type plants at ZT0 were set to 1.
(C) SOC1 mRNA levels in pSUC2::ABF plants and pFD::ABF plants. Note that SOC1 levels increased in pSUC2::ABF3 and pSUC2::ABF4 plants compared with wild-type plants. For qPCR, data obtained from three biological replicates are shown. Asterisks represent significant differences derived from one-way ANOVA followed by Dunnett's multiple comparisons tests (ns, not significant; **P ≤ 0.01, ***P ≤ 0.001).
(D) Flowering time of pSUC2::ABF3 soc1-2 plants at 23°C under LD conditions. Asterisks represent significant differences derived from two-tailed Student's t-tests (**P ≤ 0.01, ****P ≤ ).
(E and F) Yeast one-hybrid assays of the interaction of AD-ABF3 and AD-ABF4 with the pHIS2-SOC1 promoter (E). AD, Gal4 activation domain fusion; BD, Gal4 DNA-binding domain fusion. Positive control (PC): pNYE1-pHIS2; negative control (NC): empty pHIS2 vector. (F) ChIP–qPCR assays of HA-ABF3 and HA-ABF4 binding to SOC1 promoter regions using pABF3::3×HA:ABF3 abf3 and pABF4::3×HA:ABF4 abf4 plants. The numbers indicate the distance from the translational start site of SOC1. Region VII and TUBULIN (TUB) were used as negative controls. Fold enrichment of TUB was set to 1. For qPCR, data obtained from three biological replicates are shown.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Author Terms and Conditions

7. Figure 6 ABF3 and ABF4 Interact with NF-YC3, NF-YC-4, and NF-YC9.
(A) Sequence of region IV in the SOC1 promoter. Note that the NF-Y-binding element (NFYBE; bold type) is present in this region.
(B) Yeast two-hybrid assays of the interaction of AD-ABF3 and AD-ABF4 with BD-NF-YC3, BD-NF-YC4, and BC-NF-YC9. AD, Gal4 activation domain fusion; BD, Gal4 DNA-binding domain fusion. Positive control (PC): AD-T (SV40 large T-antigen) and BD-p53 (murine p53); negative control (NC): AD-T and BD-Lam (Lamin).
(C) Co-immunoprecipitation assays using Arabidopsis protoplasts. Western blots probed with anti-HA and anti-T7 antibodies showing that ABF3 and ABF4 co-immunoprecipitate with NF-YC3 (left), NF-YC4 (middle), and NF-YC9 (right).
(D) BiFC assays showing that ABF3/4 interacts with NF-YC3/4 in the nucleus (arrowhead) of N. benthamiana epidermal cells. BF, bright field. Scale bars, 100 μm.
(E) Expression levels of SOC1 in abf3 abf4, abf2 abf3 abf4, nf-yc3 yc4 yc9, and co-101 mutants with or without ABA treatment. SOC1 expression levels without ABA treatment in each line were set to 1.
(F) GUS-staining of 5-day-old pSOC1::GUS plants carrying an intact NFYBE or a mutated NFYBE (mNFYBE) 8 h after 20 μM ABA treatment. NFYBE and mNFYBE sequences are shown in the upper panel. Photos of a whole seedling and a magnified view of a cotyledon are shown in the lower panel.
(G) SOC1 and GUS mRNA levels in the cotyledons of pSOC1::GUS plants carrying an intact NFYBE or an mNFYBE. For qPCR, data obtained from three biological replicates are shown.
Asterisks represent significant differences derived from two-tailed Student's t-tests (ns, not significant; *P ≤ 0.05, **P ≤ 0.01).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Author Terms and Conditions

8. Figure 7
Reduced Drought-Escape Response in abf3 abf4, abf2 abf3 abf4, soc1, and nf-yc3 yc4 yc9 Mutants.
(A) Increase in DREB2A and DREB2B mRNA levels in drought-treated Col-0, abf3 abf4, abf2 abf3 abf4, soc1-2, and nf-yc3 yc4 yc9 plants.
(B and C) Total leaf numbers (B) and morphology (C) of Col-0, abf3 abf4, abf2 abf3 abf4, soc1-2, and nf-yc3 nf-yc4 nf-yc9 mutants with or without drought treatment.
(D) Expression levels of SOC1 in Col-0, abf3 abf4, abf2 abf3 abf4, and nf-yc3 yc4 yc9 mutants with or without drought treatment. Note that soc1-2 mutants were excluded from this analysis because soc1-2 mutants are RNA-null. For qPCR, data obtained from three biological replicates are shown.
(E) A working model of ABF–NF-Y complex function in ABA- or drought-responsive flowering. Drought and ABA induce the expression and activity of ABF3 and ABF4. ABF3/4, together with their interacting partner, the NF-Y complex, bind to the NFYBE region of the SOC1 promoter and promote its expression to accelerate flowering.
Asterisks represent significant differences derived from two-tailed Student's t-tests (A and D) and one-way ANOVA followed by Dunnett's multiple comparisons tests (B) (ns, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ ).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Author Terms and Conditions